This invention relates to vaccine against a rickettsial disease called heartwater, to a rickettsia used in the preparation of said vaccine, to a method of producing a said vaccine, and to a method of vaccinating mammals and, in particular, domestic and wild ruminants.
Rickettsiae are non-motile bacteria which depend on the intracellular milieu of host cells for growth and are capable of infecting mononuclear and endothelial cells. As a result, they were initially considered to occupy a special taxonomic niche between viruses and bacteria but are now classified within the bacteria.
Rickettsiae are transmitted to mammals by arthropod vectors such as lice, ticks, and mites. They are responsible for a variety of diseases including typhus, Rocky Mountain spotted fever, and rickettsial pox. In southern Africa, tick bite fever and heartwater are common rickettsial diseases. As a result of their dependence on the intracellular milieu of their host cells for growth, it is difficult to treat rickettsial infections. To enable a host mammal to develop immunity against this infection, bacteriostatic antibiotics are used. These include the tetracycline group of antibiotics and the chloramphenicols.
Heartwater is an economically important disease of domestic ruminants caused by the rickettsia Cowdria ruminantium and transmitted by Amblyomma tick species. Heartwater is a constraint to livestock production because it can cause mortality rates of 20-80% in susceptible animals.
Currently, there is only one method of vaccination of domestic animals against heartwater. This method involves infection of the animal intravenously with live virulent Cowdria ruminantium rickettsiae followed by treatment with tetracycline antibiotics when clinical disease is manifested. The treated animal then develops immunity to heartwater. However, if treatment is too early the animal fails to become immunized and, if it is treated too late, death can occur. This method of vaccination is both laborious and expensive and is accompanied by considerable risk and uncertainty. Hence a vaccine that is easy to administer, is inactivated and induces protection against mortality would have a major impact on livestock production in areas of the world that are affected by this disease, namely sub-Saharan Africa and the eastern Caribbean. Such a vaccine would have wide-scale application if it did not require stringent handling, transportation, and storage conditions.
The subject invention provides materials and methods for preventing, or reducing the severity of, heartwater. In accordance with this invention, there is provided a vaccine against heartwater comprising cultured inactivated Cowdria ruminantium rickettsiae mixed with a suitable adjuvant. The vaccine of the subject invention can be injected into an animal and, once injected, induces immune responses which protect the animal from severe heartwater when exposed to infection with Cowdria ruminantium rickettsiae.
In a preferred embodiment of the subject invention, there is further provided for the rickettsia to be the Mbizi strain of Cowdria ruminantium. The rickettsia of the subject invention was deposited with the National Bank for Industrial Micro-organisms and Cell Culture in Sofia, Bulgaria (NBIMCC) located at 125, Tsarigradskochausse Blvd., Block 2, 1113 Sofia, Bulgaria under accession number 3568 on Nov. 2, 1998.
In a specific embodiment, the rickettsia are cultured, using methods known in the art, in bovine endothelial cells and harvested from the cultured supernate by centrifugation. The harvested rickettsiae are inactivated by suspension in a solution of xcex2-propiolactone in phosphate buffered saline at a temperature of 4xc2x0 C. for a period of 2.5 hours. The inactivated rickettsiae can be stored frozen at a temperature of between xe2x88x9240xc2x0 C. to xe2x88x9280xc2x0 C., prior to mixing with an adjuvant.
In a preferred embodiment, the pathologically inactivated rickettsiae are mixed with an adjuvant. The concentration of rickettsiae is approximately 300 xcexcg protein per milliliter of phosphate buffered saline mixed with about 1 ml of adjuvant. The vaccine may be stored on ice until ready for use. The vaccine can be injected, subcutaneously, preferably in the shoulder region of the animal.
The invention further extends to a method of producing a vaccine for use against heartwater. In a specific embodiment, this method comprises the steps of:
(a) isolating and culturing pathologically active rickettsiae in a suitable culture medium;
(b) harvesting the rickettsiae from the culture medium once the concentration of rickettsiae in said culture medium has reached a predetermined minimum or until the happening of a predetermined event;
(c) rendering the rickettsiae pathologically inactive; and
(d) mixing the pathologically inactive rickettsiae with a suitable adjuvant.
The invention also provides for a method of immunizing mammals against heartwater comprising inoculating an animal, preferably cattle, alternatively sheep, goats, deer, springbok and antelope, further alternatively other susceptible ruminants, subcutaneously, preferably in the shoulder region, with a vaccine as described above and reinoculating the animal after the first inoculation, preferably between 4 and 8 weeks after the first inoculation.
The subject invention provides materials and methods for inducing a protective immune response against heartwater. In accordance with this invention, there is provided a vaccine against heartwater comprising cultured inactivated Cowdria ruminantium rickettsiae mixed with a suitable adjuvant. The vaccine of the subject invention can be injected into an animal and, once injected, induces immune responses which protect the animal from severe heartwater when exposed to infection with Cowdria ruminantium rickettsiae.
Specifically exemplified herein is the use of the Mbizi strain of Cowdria ruminantium. Preferably, the Mbizi strain is formulated with an appropriate adjuvant and then used as a vaccine composition.
The Mbizi strain of Cowdria ruminantium accession No. 3568, has been deposited for the purposes of this patent application under conditions that assure that access to this culture is available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposit will be available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.
Further, the subject culture deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of a deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it.
In a specific embodiment, the rickettsiae are cultured, using methods known in the art, in bovine endothelial cells and harvested from the cultured supernate by centrifugation. The pelletised organisms are then resuspended and washed in phosphate buffered saline after which they are rendered pathologically inactive by washing for 2.5 hours in a 0.4% by volume of xcex2-propiolactone in phosphate buffered saline at 4xc2x0 C. After inactivation the rickettsiae can be frozen to between xe2x88x9240xc2x0 C. and xe2x88x9280xc2x0 C. Either prior to freezing or after freezing, the protein content of the harvested organisms can be determined using the Lowry method.
When its use is required, the vaccine is prepared by mixing the thawed organisms with phosphate buffered saline to give a dosage unit of about 300 xcexcg protein per ml phosphate buffered saline. The vaccine may be stored in this form on ice and it can be injected subcutaneously over the shoulder area of the animal. A booster injection can be administered in the same region between about 4 and 8 weeks after the initial inoculation. The injected animal is considered to be immune to heartwater after a further 4 weeks have elapsed.
Sheep, Inoculations and Monitoring.
Merino or Merino-Dorper-cross sheep (6 months old) were used in vaccine trials. These sheep were obtained from heartwater-free farms in Ruwa and Mazowe in the regions of the highveld of Zimbabwe, where both Amblyomma tick vectors and heartwater have not been recorded since the start of veterinary surveillance around the turn of the century. Although they were free of heartwater, some sheep were serologically positive (false positives) on C. ruminantium antigen immunoblots due to cross-reactions with agents such as Ehrlichia species. It has previously been shown that such sheep are fully susceptible to heartwater challenge. To avoid any bias, such false positive sheep were distributed equally into vaccinated and control groups. The vaccinated groups were inoculated with the inactivated organisms with adjuvant and the control groups with adjuvant mixed with phosphate buffered saline (PBS; NaH2PO42H2O, 0.0028 M; Na2HPO4; 0.0072M; NaCl, 0.15 M; pH 7.3), except in the adjuvant selection trial described below. All inoculations were performed by the subcutaneous route, and any reaction at the injection site was recorded. In addition, any clinical reaction following vaccination was also recorded. Following challenge with a lethal dose of C. ruminantium (intravenous or via ticks), the rectal temperature of each sheep was recorded daily, and protection was determined by comparing differences in rickettsemia, time to death, and mortality rates between the vaccinated and control sheep. However, the ultimate indicator of protection was the level of mortality in the vaccinated compared to control groups. Clinical signs, though recorded, were not used as a parameter of protection since they are not specific for heartwater and can vary widely from peracute to mild forms of the disease.
Preparation of inactivated C. ruminantium Organisms for Vaccination
The Mbizi strain of C. ruminantium was inactivated was xcex2-propiolactone as described above. The inactivated organisms were quantified by staining with acridine orange, and by the Lowry method of protein estimation, and stored at xe2x88x9240xc2x0 C. for use in vaccine trials.